Broad-spectrum antimicrobial activity of quaternary ammonium surfactants (QAS) makes them attractive and cheap topical prophylactic options for sexually transmitted infections and perinatal vertically transmitted urogenital infections. Although attributed to their high affinity for biological membranes, the mechanisms behind QAS microbicidal activity are not fully understood. We evaluated how QAS structure affects antimicrobial activity and whether this can be exploited for use in prophylaxis of bacterial infections. Acute toxicity of QAS to in vitro models of human epithelial cells and bacteria were compared to identify selective and potent bactericidal agents. Bacterial cell viability, membrane integrity, cell cycle and metabolism were evaluated to establish the mechanisms involved in selective toxicity of QAS. QAS toxicity normalized relative to surfactant critical micelle concentration showed n-dodecylpyridinium bromide (C₁₂PB) to be the most effective, with a therapeutic index of ∼10 for an MDR strain of Escherichia coli and >20 for Neisseria gonorrhoeae after 1 h of exposure. Three modes of QAS antibacterial action were identified: impairment of bacterial energetics and cell division at low concentrations; membrane permeabilization and electron transport inhibition at intermediate doses; and disruption of bacterial membranes and cell lysis at concentrations close to the critical micelle concentration. In contrast, toxicity to mammalian cells occurs at higher concentrations and, as we previously reported, results primarily from mitochondrial dysfunction and apoptotic cell death. Our data show that short chain (C₁₂) n-alkyl pyridinium bromides have a sufficiently large therapeutic window to be good microbicide candidates.